PC-AssayDescription ::= {
aid {
id 652017,
version 1
},
aid-source db {
name "The Scripps Research Institute Molecular Screening Center",
source-id str "BRM_ACT_LUMI_1536_1X%ACT PRUN"
},
name "Luminescence-based cell-based primary high throughput screening assay
to identify activators of the function of SWI/SNF related, matrix associated,
actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM)",
description {
"Source (MLPCN Center Name): The Scripps Research Institute Molecular
Screening Center",
"Affiliation: The Scripps Research Institute, TSRI",
"Assay Provider: David Reisman, University of Florida",
"Network: Molecular Library Probe Production Centers Network (MLPCN)",
"Grant Proposal Number 1R03DA028854-01",
"Grant Proposal PI: David Reisman",
"External Assay ID: BRM_ACT_LUMI_1536_1X%ACT PRUN",
"",
"Name: Luminescence-based cell-based primary high throughput screening
assay to identify activators of the function of SWI/SNF related, matrix
associated, actin dependent regulator of chromatin, subfamily a, member 2
(SMARCA2, BRM).",
"",
"Description:",
"",
"The protein encoded by the BRM gene (SMARCA2, SNF2, SWI2) is a member of
the SWI/SNF family of proteins and is highly similar to the brahma protein of
Drosophila (1). Members of this family have helicase and ATPase activities
and are thought to regulate transcription of certain genes by altering the
chromatin structure around those genes (2). The encoded protein is part of
the large ATP-dependent chromatin remodeling complex SNF/SWI, which is
required for transcriptional activation of genes normally repressed by
chromatin. BRM is an epigenetically suppressed anti-cancer gene, which is
silenced in wide variety of solid tumors (2). Because BRM function is key for
growth control, restoring its expression routinely inhibits cancer cell
growth. For this reason, restoring BRM has potential as an anticancer
therapeutic modality (3-4). Its expression prevents cancer development as
seen in murine models system where BRM loss potentiates cancer development
10-fold (5). It is known from preliminary studies that histone deacetylase
(HDAC) inhibitors are found to restore BRM expression in cancer cell lines,
but not its function (2). However, BRM is involved in many pathways and
required by numerous transcription factors which control development,
differentiation, DNA repair, adhesion, and growth control (6). As such, the
impact of inactivating BRM and/or restoring its expression goes well beyond
growth control (7).",
"",
"Although pan-histone deacetylase (HDAC) inhibitors are found to restore
BRM expression, but not its function in cancer cell lines, specific
inhibitors of either HDAC3 or HDAC9, as well as the transcription factor
GATA3 and/or MEF2D, induce a functional BRM. These other constituents
involved in BRM regulation may also be the molecular targets. However, since
HDAC9 and GATA3 are highly overexpressed and given the limited scope of
expression of HDAC9, these proteins would be preferred targets. The fact that
each are highly over-expressed is akin to EGFR in lung cancer or HER2 in
breast cancer. Candidate compounds that are positive on the primary screen
will be re-screened using our counter-screen, where BRM has been suppressed
using anti-BRM shRNA (MG2-KDM). We have found that even the most potent
inducers of BRM, are blocked by at least 50% or more by this assay. Hence,
false positives will yield readouts of luciferase activity that show >50%
inductions, closely approximating the levels found in the primary BRM (MG213)
screen and will not have inductions <50% as observed with essentially all
BRM-specific inducers.",
"",
"Compounds will be then be verified as positive hits by a series of assays
beginning with a third confirmatory (dose response) screening. Following the
dose response screening, hits will be screened by directly measuring BRM
induction by qPCR since the BRM gene is controlled by transcription.
Additionally, we will determine the relative specificity the hits identified
by screening each for its potential to induce a number (~10) of BRM-dependent
genes. Since each of these BRM-dependent genes are controlled by different
signal transduction pathways, interference in one or more cellular pathways
(due to lack of specificity for BRM) will be demonstrated by a lack or
reduced induction of one or more of the BRM-dependent genes. Moreover, the
level of induction observed for each BRM-dependent gene is an indirect marker
for the potency of BRM induction.",
"",
"A secondary goal of this project is to group these verified compounds
based upon their relative site of action in the pathway of BRM induction. We
will use a secondary assay to differentiate compounds affecting upstream and
downstream sections in the regulatory pathway. A possible additional screen
will then be optionally performed using cells harboring anti-HDAC2 shRNA
(MGH2KD cells). Since the deacetylation of BRM is a requirement for function
thus generating a luciferase signal in these reporter cells, only the
compound(s) that affect higher-level regulatory genes (such as MAP kinase
inhibition) will be identified with a positive result using this assay. For
those compounds having an impact lower in the pathway, we will use them to
determine if they inhibit BRN2, GATA3, HDAC3, MEF2D, or perhaps HDAC9,
thereby assisting us in determining which motifs of the compound underlie the
re-expression of BRM. After this series of screens, all hits will then
undergo a series of secondary assays to determine how well they restore BRM
expression and its function in BRM-deficient cell lines. The assay provider
will explore these secondary MOA studies in additional collaboration beyond
the scope of this CPDP.",
"",
"References:",
"",
"1. Bourachot, B., M. Yaniv, and C. Muchardt, The activity of mammalian
brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA
binding domain. Mol Cell Biol, 1999. 19(6): p. 3931-9.",
"2. Glaros, S., et al., The reversible epigenetic silencing of BRM:
implications for clinical targeted therapy. Oncogene, 2007. 26(49): p.
7058-66.",
"3. Reisman, D., S. Glaros, and E.A. Thompson, The SWI/SNF complex and
cancer. Oncogene, 2009. 28(14): p. 1653-68.",
"4. Reyes, J.C., et al., Altered control of cellular proliferation in the
absence of mammalian brahma (SNF2alpha). EMBO J, 1998. 17(23): p. 6979-91.",
"5. Liu, G., et al., Two novel BRM insertion promoter sequence variants
are associated with loss of BRM expression and lung cancer risk. Oncogene,
2011. 30(29): p. 3295-304.",
"6. Coisy-Quivy, M., et al., Role for Brm in cell growth control. Cancer
Res, 2006. 66(10): p. 5069-76.",
"7. Gramling, S., et al., Pharmacologic reversal of epigenetic silencing
of the anticancer protein BRM: a novel targeted treatment strategy. Oncogene,
2011. 30(29): p. 3289-94.",
"",
"Keywords:",
"",
"Primary, PRUN, primary screen, confirmation, dose response, BRM, SMARCA,
SMARCA2, SWI/SNF, ATPase, gene, transcription, regulator, MG213, reporter,
activate, activator, inducer, increase, lumi, luc, luminescence, luciferase,
cell, cell-based, dexamethasone, HTS, high throughput screen, 1536, Scripps,
Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening
Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN."
},
protocol {
"Assay Overview:",
"",
"The purpose of this assay is to identify compounds that act as activators
(re-activators) of BRM function. This assay relies on the fact that BRM
assists the glucocorticoid receptor (GR) in activating the MMTV promoter,
which is used to drive expression of a luciferase reporter gene (7).
Compounds active in this assay will restore BRM function, allowing the
SWI/SNF-dependent GR to induce the MMTV promoter, leading to increased
luciferase expression and well luminescence. MG213 cells are incubated in the
presence of 0.1 uM Dexamethasone for 24 hours prior to be plated in 1536-well
plates at 600 cells/well in 5 ul media with Dexamethasone using Kalypsys
dispenser. The cells are immediately pinned with 45 nL of compound, control
or DMSO, spun down, and incubated overnight at 37 C. Test compounds compound
are delivered to the plates containing the MG213 cells and dexamethasone
using pintool. Chembridge 5306793 is included as a positive control on each
plate. OneGlo luciferase reagent is used according to the manufacturer's
directions. Luminescence is measured using the Viewlux plate reader.
Compounds are tested in singlicate at a final nominal concentration of 9.1
uM. Note that the cell line used for this assay has been transduced with the
E1A gene, which produces the viral oncoprotein, E1A. This protein sequesters
Rb (and Rb2) protein thus preventing cellular growth inhibition. At lower
densities, these newly modified cells have a more linear growth rate and do
not lag when plated at low density. At higher densities (which typically
impacts normal cell growth via contact inhibition), these cells maintain
their growth rate allowing these cells to grow to higher densities. This
modified cell line allows for a greater experimental range for this assay and
improves its performance (consistency) as compared to the parental MG213
cells.",
"",
"Protocol Summary:",
"",
"The MG132 cell line was routinely cultured in T-175 sq cm flasks at 37 C
and 95% relative humidity (RH). The growth media consisted of RPMI -1640
supplemented with 5% v/v certified fetal bovine serum, 500 ug/mL Geneticin,
and 1X antibiotic mix (penicillin, streptomycin, and neomycin).",
"",
"Prior to the start of the assay 600 cells in a 5 ul volume of assay media
(growth media as above except without geneticin and with 2.5% FBS and 0.1uM
Dexamethasone) were dispensed into each well of 1536-well tissue
culture-treated microtiter plates. The assay was started immediately by
dispensing 45 nL of test compound in DMSO (0.69 % final DMSO concentration),
DMSO alone, or CB5306793 to the appropriate wells. Next, the plates were
incubated for 24 hours at 37 C (5% CO2, 95% RH). The assay was stopped by
dispensing 5 ul of One Glo luciferase substrate to each well, followed by
incubation at room temperature for 15 minutes. Well luminescence was measured
on the ViewLux plate reader.",
"",
"The percent activation for each compound was calculated as follows:",
"",
"%Activation = ( 1 - ( ( Test_Compound - Median_High_Control ) / (
Median_Low_Control - Median_High_Control ) ) ) * 100",
"",
"Where:",
"",
"Test_Compound is defined as wells containing test compound.",
"Low_Control is defined as wells containing DMSO.",
"High_Control is defined as wells containing CB5306793.",
"",
"PubChem Activity Outcome and Score:",
"",
"A mathematical algorithm was used to determine nominally activating
compounds in the Primary Screen. Two values were calculated for each assay
plate: (1) the average percent activation of test compound wells and (2)
three times their standard deviation. The sum of these two values was used as
a cutoff parameter for each plate, i.e. any compound that exhibited greater %
activation than that particular plate's cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% of the
highest observed primary activation value. Negative % activation values are
reported as activity score zero.",
"",
"The reported PubChem Activity Score has been normalized to 100% observed
primary activation. Negative % activation values are reported as activity
score zero.",
"",
"The PubChem Activity Score range for active compounds is 100-6, and for
inactive compounds 6-0.",
"",
"List of Reagents:",
"",
"MG132 cell line (provided by Assay Provider)",
"RPMI-1640 medium (Invitrogen, 11875-119",
"Geneticin (Invitrogen, part 10131-035)",
"100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)",
"Trypsin-EDTA solution (Invitrogen, part 25200-056)",
"Fetal Bovine Serum (Invitrogen, part 16000-044)",
"One Glo Assay Kit (Promega, part E6130/QTE30675 )",
"CB5306793, Chembridge",
"T-175 tissue culture flasks (Corning, part 431080)",
"1536-well plates (Corning, part 789173)"
},
comment {
"Due to the increasing size of the MLPCN compound library, this assay may
have been run as two or more separate campaigns, each campaign testing a
unique set of compounds. In this case the results of each separate campaign
were assigned ""Active/Inactive"" status based upon that campaign's specific
compound activity cutoff value. All data reported were normalized on a
per-plate basis. Possible artifacts of this assay can include, but are not
limited to: dust or lint located in or on wells of the microtiter plate,
compounds that non-specifically modulate luciferase activity, and compounds
that quench or emit luminescence within the well. All test compound
concentrations reported are nominal; the specific concentration for a
particular test compound may vary based upon the actual sample provided by
the MLSMR."
},
xref {
{
xref gene 6595,
comment "SMARCA2 SWI/SNF related, matrix associated, actin dependent
regulator of chromatin, subfamily a, member 2"
},
{
xref mim 600014
},
{
xref dburl "http://mlpcn.florida.scripps.edu/"
},
{
xref taxonomy 9606
},
{
xref pmid 10330133,
comment "Reference (1)"
},
{
xref pmid 17546055,
comment "Reference (2)"
},
{
xref pmid 19234488,
comment "Reference (3)"
},
{
xref pmid 9843504,
comment "Reference (4)"
},
{
xref pmid 21478907,
comment "Reference (5)"
},
{
xref pmid 16707429,
comment "Reference (6)"
},
{
xref pmid 21478905,
comment "Reference (7)"
}
},
results {
{
tid 1,
name "Activation at 9.1 uM",
description {
"Normalized percent activation of the primary screen at a compound
concentration of 9.1 micromolar."
},
type float,
unit percent,
tc {
concentration { 910000038146973, 10, -14 },
unit um
}
}
},
revision 1,
target {
{
name "SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 2, isoform CRA_a [Homo sapiens]",
mol-id 119579215,
molecule-type protein,
organism {
org {
taxname "Homo sapiens",
common "human",
db {
{
db "taxon",
tag id 9606
}
}
}
}
}
},
activity-outcome-method screening,
grant-number {
"1R03DA028854-01"
},
project-category mlpcn
}